Replacing MPS1 T675 and T676 with aspartic acids restores MPS1 kinase activity and checkpoint functionality. (A) GFP-MPS1^WT, MPS1^KD, MPS1^AA, or MPS1^DD cells were arrested in mitosis. BUBR1 and CENP-C were detected using antibodies, and MPS1 by GFP fluorescence. (B and C) Bar graphs show mean kinetochore-associated GFP-MPS1 (B) and BUBR1 (C) ± SEM. (D) The mean mitotic index ± SEM of GFP-MPS1^WT (n = 2,490 cells), MPS1^KD (n = 2,451 cells), MPS1^AA (n = 2,689 cells), or MPS1^DD (n = 3,211 cells) cells after 16-h treatment with 0.33 µM nocodazole is shown. (E) MPS1 autophosphorylation was measured by Western blotting and ³²P incorporation. Equal loading of the different MPS1 forms was confirmed by Western blotting of λ-phosphatase–treated proteins. (F) Phosphorylation of GST-KNL1^728–1,200 by FLAG-MPS1 proteins was assessed by Western blotting with anti-KNL1^pT875. Equal loading was confirmed by anti-MPS1 Western blotting. (G) Cartoon illustrating how MPS1 TT675/6 mutations affect MPS1 activity.